Production of Clinical-Type Dextran PARTIAL ACID HYDROLYSIS AND FRACTIONATION FROM L. mesenteroides NRRL B-742 AND S. dextranicum NRRL B-1254 I. A. WOLFF, R. L. MELLLES, R. L. LOHYIAR, N. N. HELLMAN, S. P. ROGOVIN, P. R. WATSON, J. W. SLOAN, B. T. HOFREITER, B. E. FISHER, AND C. E. RIST .Vorthern Utilization Research Branch, Agricultural Research Service,
RECEKT publication from this laboratory ( 1 6 ) discussed the partial hydrolysis and fractionation of the dextran produced by Leuconostoc mesenteroides NRRL B-512 In view of the widespread interest in dextran as a blood plasma volume expander, for comparative purposes the preparation of clinical-size fractions from other dextrans differing in chemical structure from the NRRL B-512 polysaccharide was investigated. The selection of dextran types for study was based on their percentage of 1,6’-glucosidic linkages as determined by oxidation with sodium metaperiodate (6). The dextrans formed by L. mesenteroides NRRL B-742 (68 to 69% 1,6’-linkages) and Streptobacterium deztrunicum NRRL B-1254 (90% 1,6’-linkages) provided one sample differing by a substantial amount from the NRRL B-512 product (95% 1,6’-linkages) and another sample of intermediate type. The heterogeneity of the NRRL B-742 and B-1254 dextrans, especially the presence of approximately cqual amounts of two structurally different polysaccharides in the NRRL B-742 dextran ( 1 , 8 ) , contributed to the complexity of this work. This paper describes the preparation of clinical-type fractions fioin the NRRL B-1254 and B-742 dextrans, as well as some experiments on the hydrolysis and fractionation of the individual polysaccharide components of the latter. As before (16), the term “clinical” fraction or “clinical-type” dextran refers t o a product that meets the molecular size and size distribution requirements for dextran suitable for the preparation of clinical injection solutions (14). The yields of clinical-type dextran reported are not necessarily the maximal, since the primary objectives of this experimentation were to ascertain whether the various kinds of dextrans required different handling, to compare the depolymerization processes and products, and to accumulate sufficient laboratory data to enable preparation of several pounds of the clinical-type fraction from each of the two dextrans chosen. iMATERIALS AND METHODS
U. S. Department
210 o
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of Agriculture, Peoria, I l l .
,
,
I
I
0 33
35
37 39 41 43 95% ETHANOL, PERCENT
45
47
Figure 1. Precipitability of Native Dextrans with 95% Ethanol
materials contained negligible amounts of fructose (16)and had molecular weights of many million, as indicated by the lightscattering method. The heterogeneity of these natural dextrans as compared with the native dextran synthesized by the NRRL B-512 organism is indicated by inflections in the curves illustrating their precipitability a t different alcohol concentrations (Figure 1) The data for these graphs were obtained as follows:
DEXTRANS.The dextrans were all prepared in whole culture; they were isolated by known procedures ( 6 ) . Further data on the origin and characterization of these dextrans will be published shortly ( 3 ) . Separation of the NRRL B-742 dextran, preparaTo a 2% aqueous solution of the dextran in a 250-ml. centrifuge bottle kept in a bath a t 25’ c. was added with stirring a tion A, into two fractions was carried out in 8.25y0 aqueous solution with 43% (vol./vol.) of 95Oj, ethanol. The 45% of the starting material that was OF UNDEGRAUED DEXTRANS TABLE I. CHARACTERISTICS insoluble a t that ethanol conGlucosidic Linkages, %“ Analysis, % centration was further freed Designation of Dextran 1,6’1,4’-like 1,3’-like [q] Nitrogen Phosphorus Ash from traces of the more NRRL 3-1264, preparation C 91 8 2 0.665 0.02 0 04 soluble portion by precipitaNRRL B-1254, preparation D 89 6 5 0.484 0.06 0 : 065 0.31 NRRL B-742b, preparation A 0.05 0.07 69 18 14 0.259 0.30 tion from 2% aqueous solution Portion soluble in 43y0 ethanol 58 24 18 0.318 . . . a t 39% ethanol concentration. 0.08 Portion insoluble in 39% Each major fraction was de0.02 ethanol 81 25 ... 0.139 0.53 0.07 NRRL B-742C, preparation B 68 18 14 0.279 0.06 hydrated with methanol and a Determined b y the periodate oxidation method (6.8). dried. In Table I are listed b Corn steep liquor used as nitrogen aource in medium used for production. Yesst extract and tryptone used a8 nitrogen source in medium. analytical data on the dextrans. The purified starting
2605
INDUSTRIAL AND ENGINEERING CHEMISTRY
2606
measured volume of 95% ethanol until incipient turbidity was observed. After the solution had been stirred for an additional 5 minutes, it was centrifuged. The supernatant liquor was d e canted into another 250-ml. centrifuge bottle, and another increment of alcohol Tvas added to precipitate another fraction. The process was repeated until over 90% of the dextran had been removed. I n calculating the percentage of alcohol (vol./vol.) present in the supernatant portions, no correction was made for contraction in volume on mixing, or for any alcohol left with the precipitate. Each precipitated fraction was made up to a known volume in water, and the amount of dextran present was determined from the optical rotation or by anthrone analysis ( 11).
tion of fractions ( 1 6 ) are employed-Le., all fractions obtained in the first fractionation stage are designated by capital letters, those in the second stage by arabic numerals, and those in the third stage by lower-case letters.
r '
4.8 1.33
6 1.23
ti
Hours
Oi96
DH
I 7
i
a
I
'
L
-
16
I
17
I
SELPTIVE VISCOSITY
~
19
I
I
21
OF YYDROLYZATE
Figure 2. Relative Viscosities of 80' C. Mydrolyzates from NRRL B-742 Dextran ms. Yields of Fractions Fraction A B C D
Fiactionation Range with Q5y0 Ethanol, % At pH 1.06 f o r 7 hr. 0-45 0 -45 45-51 45-52 51-57 52-55 57-61 58-65
HYDROLYSIS ASD FRACTIOSATIOY PROCEDURES. The laboratory hydrolysis and fractionation procedures have been described ( 1 6 ) . For larger scale operation a 90-gallon, jacketed glass-lined kettle was used for the hydrolysis, which was carried out as before except that the sulfuric acid was added to the dextran solution before heating was begun. If this was not done, sufficient agitation of the viscous solution to effect good heat transfer was difficult to attain, and a gelatinous layer of dextran was deposited on the xall of the kettle. The fractionation of the larger batches was effected in 50-gallon stainless steel drums, equilibrated in a constant-temperature room maintained at 25 O C. For separation of the two liquid phases a t each stage of the fractionation of the larger batches the supernatant liquor was decanted from the precipitated sirupy portion by pumping i t off through a filter. All fractionations described in this paper vere made by the addition of 95% ethanol to an aqueous solution of the dextran. The mixtures were allowed to come to 25" C. and remain a t this temperature for a t least 1 hour before the fractions mere separated. Concentrations of 95'% ethanol are expressed as per cent by volume. The conventions previously established for designa-
L
L
_
17
-
_
-
I8
U
19
RELATIVE VISCOSITY OF HYDROLYZATE
Figure 3. Relative Viscosities of 90" 6. Hydrolyzates from NRRL B-742 Dextran ws. Yields of Fractions Fraction A B C D
01 15
Hours 1.15 p~
&--15
6.3
r 2.3
I
k j
o 5.3 Oi75
7.3 1.537
I
i
40p
Figure 1 shows that S R R L B-742 dextran, preparation A, consisted of approximately equal portions of two polysaccharides differing in alcohol precipitabilities and, as indicated in Table I, in chemical structures. The two preparations of native B-742 dextran (A and B) differed slightly in intrinsic viscosity and in alcohol precipitation curves. For XRRL B-1254 dextran the prccipitation curve indicates that 10 to 20% of the material may differ from the remainder.
Vol. 46,No. 12
Fractionation Range with 9 6 % Ethanol, % 0-45 45-51 61-57 57-64
DEXTRAN COKCENTRATION. Whenever clarit,y of the Bolut,iona permitted, the dextran concentration was determined by optical rotation in aqueous solution, using a specific rotation (at 25" C., sodium D-line) of +198" for the KRRL B-1254 dextran, +210° for the NRRL B-742 dextran, and + 2 E 0 and +200", respectively, for the more and less soluhle fractions of t.he KRKI, B-742 dextran. Anthrone analysis ( 1 1 ) was used for those solutions which were too turbid for polarimetric analysis. Such solutions were encountered with the higher molecular weight fractions only. VISCOSITT. As in the work on the NRRL B-512 dextran, inherent viscosity (In qrei./c, designated ( q 1 ) was found to be virtually the same as the intrinsic viscosity of the depolymerized dextran fractions: and was therefore used routinely. Viscosity measurements were carried out as previously described ( 1 6 ) ,and the same terminology is used as before. MOLECULAR WEIGHT. Number-average molecular weights of depolymerized dextran fractions were determined by the Soniogyi reducing-power procedure (2, 16). Weight-average molccular weights were determined by the light-scattering procedure by B. L. Lamberts and R. Tobin of the Analytical, Physical-Chemical, and Physics Section of this laboratory. CLINICAL-TYPE FRACTIONS FROM KRRL 8-742 DEXTKAN
PREPARATIOS A. It was found by trial that a hydrolyzed dextran fraction thrice fractionated between 45 and 51% of 95% ethanol had the desired molecular weight ( 1 4 ) . 'L'he effect of different conditions and extent, of hydrolysis on thc amount fraction (fraction R ) produced in the first of the 45 to fractionation of the hydrolyzate is depicted in Figures 2 and 3. Included for comparison are the other fractions obt,ained. At 80" C. (Figure 2) the yields of 45 to Sly0 and 51 to 5 i % fractions did not vary greatly when the relative flow time (hereafter for convenienre called relative viscosity) of the hydrolysis mixture at the termination of the hydrolysis was between 1.5 and 2.1. There remained a large amount of high molecular weight material (0 to 45% fraction). Degradation of this mat,erial a t 80" C. would have required a longer time or a higher acid
December 1954
INDUSTRIAL AND ENGINEERING CHEMISTRY
2607
concentration than i t was desirable t o use. When high acidity TABLE 11. CHARACTERISTICS OF B-2-b FRACTIONS combined with a long reaction time (7-hour hydrolysis a t p H 0.77) ( CLINICAGTYPE) was used to cause breakdown of this high molecular weight mateB-742 B-1254 (Preparation B) (Preparation rial, the yield of very low molecular weight material (57 t o 6401, Mw (li ht scattering) 6L,OOO 87,500 fraction) increased while the yield of the desired fraction was iMw offowest 5-10% 30, OOOa 2 9 , 300a nearly unchanged. IMW of highest 5-10% 100,000= 165,OOOb M N (Somogyi method) 31,400 41,800 At 90 O C. (Figure 3), when over-all hydrolysis was carried out Intrinsic viscosity 0.153 0.229 Intrinsic viscosity of lowest &IO% 0.126 0.167 t o approximately the same relative viscosity as at 80’ C., the Intrinsic viscosity of highest 5-1070 0.157 0 e54 0 to 45% fraction was degraded t o a large extent and this degra[ala: i n water +2100 + 1980 p H of 6% aqueous solution 6.4 6.0 dation resulted in increased quantities of 45 t o 51% and 51 t o Buffering capacity (as ml. of 0.1N 57% fractions. The maximum amount of 45 to 51% fraction was NaOH/I. of 6% solution) 0.4 0.4 Nitrogen, % 0.01 0.01 obtained at relative viscosities between 1.50 and 1.85. Ash, % 0.08 0.06 Heavy metals, based on dextran I n the case of the N R R L B-512 dextran (16) the extent of (as Pb), p.p.m. 12 13 hydrolysis appeared to be the dominant factor controlling the Apparent fructose, % 0.05 0.02 Glucosidic linkage (by periodate distribution of hydrolysis products; the exact conditions of analysis), % ’ 1,6’70 91 hydrolysis were of lesser importance. B y contrast, consideration 1,4’-like 13 6 of the data presented in Figures 2 and 3 indicates t h a t the 1,3’-like 18 3 conditions chosen for hydrolysis can definitely be important in a 8% of total fraction. b 12% of total fraction. controlling the distribution of products in the N R R L B-742 dextran hydrolyzates. Thus, for example, when the relative viscosity of the final hydrolyzate was near 1.7, the yield of 0 to merized N R R L B-512 fraction (16). The alcohol-fractiona45% fraction varied from 48% for the hydrolysis carried out a t tion range required for the isolation of clinical-type dextran 80 O C. to 21 % for the 90 O C. hydrolysis, and the respective yields is markedly different for the two polysaccharides, the less for the 45 t o 51% fraction were 22 and 34%. alcohol-soluble material resembling the N R R L B-512 dextran In other hydrolyses carried out a t the same temperature and t o hydrolysates in this respect. These data re-emphasize t h e the same viscosity end point, variations in the distribution of importance of the chemical structure of a dextran in governfractions have been obtained when different combinations of ing the conditions for isolation of clinical-size fractions as well a s hydrolysis time and acid concentrations were used. As shown in the properties of such depolymerized fractions. The variability previous studies on B-512 dextran the viscosity of a fraction in yield of clinical-type fraction obtained from the native (unisolated between given limits of alcohol concentration is defractionated) N R R L B-742 dextran with conditions of hydrolysis pendent on the composition of the hydrolyzate fractionated. could easily be due to different extents of attack of the acid on the The greater dependence of the character of the hydrolyzate component polysaccharides under the particular set of hydrolysis on the conditions of hydrolysis for the N R R L B-742 dextran may conditions chosen. I n fact, the N R R L B-742 clinical-type fracprobably be ascribed to its heterogeneity and t o complexities tion described in Table I1 would appear, on the basis of the prcresulting from differences in behavior of the component polyportion of different types of glucosidic linkages it contains, t o be saccharides in both the hydrolysis and the fractionation steps derived largely from a polysaccharide resembling the 43% of the procedure. ethanol-soluble portion of N R R L B-742 dextran, preparation A PREPARATION B. T o obtain a sufficient quantity of clinical(Table 111). None of the clinical-type fractions showed a n intype N R R L B-742 dextran for bottling, 46.3 pounds of preparaflection in its precipitation curve like that of the native N R R L tion B was hydrolyzed a t 80” C. in 6% solution a t p H 1.04, using B-742 dextrans (Figure 1). sulfuric acid, t o a relative viscosity (measured a t 25” C., corrected t o 5% dextran concentration) of 2.07. Seven hours and 38 H Y D R O L Y S I S S T U D I E S OF N R R L B-1254 DEXTRAN minutes was required. T h e neutralized hydrolyzate was fractionated as shown in Figure 4, t o yield 18.1% of product having PREPARATION OF CLINICAI~TYPE FRACTIONS. Initial hydrolyses of N R R L B-1254 dextran, preparation C, indicated t h a t this the properties shown in Table 11. This clinical-type fraction met all of the currently specified chemical requirements for dextran dextran was similar t o the N R R L B-512 dextran. A clinical-type fraction ( 1 4 ) was precipitated from a hydrolyzate ( S O o C., p H 1, for injection ( 1 4 ) except those pertaining t o viscosity. The innate low viscosity of this dextran for a given molecular weight sulfuric acid, 5% dextran solution, final relative viscosity 2.4) by makes simultaneous conformity to both the molecular weight and three fractionations between 40 and 46% of 95% ethanol. viscosity specifications difficult, if not impossible, to attain since Approximate relationships between inherent viscosity and the specifications pertain t o a “B-512-like” dextran. The values molecular weights of fractions obtained in this manner were shown in Table I1 were the same for the clinical-type fraction M.@JJ~~ ( 7 1 = 4.25 X M w ~and . ~( 7 ~ )= ~ 8.67 X originally obtained as for a portion isolated from the bottled, sterile injection solution (containing 0.9% sodium chloride) Four hydrolyses illustrating the effect of variation in temperature and extent of hydrolysis on the yield of clinical-type fraction from prepared from it. COMPONENTPOLYSACCHARIDES OF PREPARATION 9.The more and less alcohol-soluble TABLE 111. PROPERTIES OF CLINICAL-TYPE FRACTIONS FROM HYDROLYZATES OF COMPONENT components of N R R L B-742 POLYSACCHARIDES OF N R R L B-742 DEXTRAN, PREPARATION A dextran were different from each other, as illustrated by Final Relative Fractionation Inherent the data of Table I11 which PolyViscosity Range with B-2-b or ’ Glucosidic-Linkages, % saccharides, of 95% Fraction, Intrinsic depict the properties of clinicalHydrolyzed Hydrolyzate Ethanol % Viscosity MN iMw 1,6‘- 1,4’-like 1,3’-like type fractions obtained from Portion soluble hydrolyzates of these matein 4376 ethanol 1.99 45-51 14 0.137 40,000 57,600 64 15 22 rials. Both the clinical-type Portion insoluble in 39% fractions have low viscosities ethanol 1.99 40-46 15 0.135 41,400 57,900 81 24 ... 1.79 40-46 15 0.132 36,450 ... 83 22 for their molecular weights as compared with a depoly-
...
INDUSTRIAL A N D E N G I N E E R I N G CHEMISTRY
2608
Neutral Hydrolyzate of B-742 Dextran, Preparation B (46.3 lb. in 5 % aqueous solution) added 95% ethanol to 45% concentration
Precipitates
I
j.
_ _ _ _ . - _ _ - ~ ~~~ ~
.1
I
Fraction A (18.0 lb., 38.9% yield)
28.3 lb., 61.1% yield to 51% ethanol
II
Fraction R (13.5 lb., 29.2% yield) to 570 concn. in miter to 45% ethanol
13.9 lb., 30.0% yield t o 5T7, ethanol
I Fraction C ( 5 . 3 1b.j 11 440yield)
._
II
Fraction B-l(O.ObOyield, trace)
Fraction D (8.6 lb.. 18 6% 7 icltl)
I
13.41b., 2 k 9 % yield to 51% ethanol
'~
I
Fraction B-2 (10.5 lb., 22.7% yield) to 6 % concn. in vater, supercentrifuged, and protein residue discarded; centrifugate diluted t o 5% concn. 10 3 lb., 22 2% yield to Gi% ethanol
li
Supernatant.:
_ _ ~ ~ _ _ ~ ~ ~ ~ ~ ~ _ _ _ _ _
~
__
ii
Fmctioii B-2-a
Vol. 46, No. 12
Fraction B-3 (2.9 lb., 6 3% J ield)
(O.Oyo> icltl, tiace?
to 51 %'ethitllol
I'
Fraction B-2-b (8.4 lb., 18.1% yield)
Fraction B-2-l(1.9 lb., 4.17, jield)
Final Clinical-Type Fraction Figure 4.
Fractionation Scheme for NRRL H-742, Preparation B, Dextran Hydrolyzate
preparation C of the B-1254 dextran are included in Table IT'. Hydrolysis to a relative viscosity of 2.4 a t 80' C. resulted in the best yield of clinical-type fraction. The effect of temperature was less important than was the extent of hydrolysis. Hydrolysis and fractionation of 37 pounds of preparat'ion D (Table IT') for product,ion of a sufficient quantity of dextran for making clinical injection solutions closely pa.ralleled laboratorjresults on preparat,ion C xTith the exception t h a t it was necessary to precipitate the clinical-size fract,ion between 39 and 457, of 95% ethanol. ISOLATIOS OF FRACTIONS WITH Low PERCESTAGE OF 1,6'LIXKAGES. A 5% solution of KRRL B-1254 dextran, preparation C, was hydrolyzed in sulfuric acid a t p H 2.5 and 100" C . to a relative viscosity of 8.5. This hydrolyzate was separated into four major fractions by fractional precipitation with 95% ethanol, and each major portion was subfractionated t o give a total of 40 subfractions varying in inherent viscosity from 0.72 t o 0.10. Surprisingly, the first three, which were precipitated a t the lowest alcohol percentages, had only 70 to 23% of 1,6'-glucosidic linkages as compared with over 90% 1,6'-linkages in each of the other
%.el.
Preparat,ion
c
C
C C
D
D a
M w by
P 13
(H2801) 1.03 0.95 1.03
0.95 1.03 1.04
a t E n d of Hydrolysis 2.40 3.10 3.05 2.47 2.41 2.40
light mattering W&Q 40,600.
Temp.,
C. 80 70 80 70 80 80
O
fractions. These three fractions, represent,ing about 5% of tlic original dext,ran, had ion-er inherent viscosities (0.35 t o 0.44) th did succeeding subfractions which decreased progressively 111 viscosity beginning with the fourth subfraction ( { ] = 0.72). Subfractions 1 t o 3, with their low percentages of 1,6'-linkagcs, may represent, essentially unchanged, that portion of the nativo dextran responsible for t.he anomaly in the lower port,ioii of its precipitation curve (Figure 1). The first subfraction was fount1 to have a weight-average molecular weight of I .6 x' lo7, ensuring that its lower viscosity was not caused by extensive degradation. Thus, the fractionation of this slight,ly hydrolyzed NRRL 13-1254 dextran occurred according t,o molecular type as well as molccu1:tr size. KINETICS OF HYDROLYSIS
Conditions and procedures chosen for measurement of tlic hydrolysis rates were the same as those previously used for the N R R L R-512 dextran ( 1 6 ) . Data on hydrolysis of the dextrans studied, all of JThich were consistent with considerat,ion of t,hc hydrolyses as first-order reactions. are summarized in Table V.
Fractionation Range w i t h 957, Ethanol
10-46 40-46 40-46 40-46 39-45 40-46
A 17 38 38 18 10 26
B 60
35 39 48 50 42
C 33 27 25 34 38 32
Yield of Fractions, 70 B-1 B-3 B-2-8 B-2-0 1 11 3 5 1 8 1 6 3 7 5 2 0 10 7 5 0 1G 0 0 0 9 0 9
B-2-b 30 20 21 20 20
24
Properties of H-2-13 Fraction 171 M,v
0.213 0 I220
0,213
0.213 0,229 0.187
34,300 38,600 34,800 37,700 41,800 2 6 , OOOa
INDUSTRIAL AND ENGINEERING CHEMISTRY
December 1954
Measurements were made a t total extents of hydrolysis of from 2 to 5%. The specific reaction rate constants for the N R R L B-1254 and N R R L B-742 dextrans were larger than that of the K R R L B-512 dextran. This observation is consistent with the reportedly greater resistance t o acid hydrolysis of the a-1,6’glucosidic linkage as compared with the ~1,4’-linkage( 4 , 13, 1 7 ) . The rate constants for a-l,3’-linkages and au-l.2’-linkages which may also be present in some of the dextrans, are not yet reported. The temperature coefficients of hydrolysis and the energies of activation for the NRRL B-742 and B-1254 dextrans do not differ markedly from those for the N R R L B-512 dextran.
TABLE V. KINETICS OF I~YDROLYSIS OF DEXTRANS (Siilfuric acid, pH 1, 5 % dextran concentration) First-Order Reaction R a t e Constant, Hr.-‘ X 103 ~ ~ Dextran 80’ C. 70” C. cient 3 . 8 1 0.911 4.0 N R R L B-512a 4 3 N R R L B-1254, preparation C 5.19 1.21 3.9 N R R L B-742, preparation A 7.00 1.82
~ $ , io^^
Cal./Mole 33,100 35,100 32,400
MATHEMATICAL CONSIDERATION OF HYDROLYSIS
Kuhn ( 7 ) has shown t h a t in the hydrolytic cleavage of a very large linear polymer, all of the bonds of which are equivalent, the maximum yield of a degraded fragment is given by the expression
2609
polymerization of clinical-type dextran fractions. Curves B and C represent the theoretical distributions for the respective extents of hydrolysis (2.3 and 4.5%) attained in hydrolyzates of K R R L B-1254 and B-742 dextrans a t the stage required for isolation of good yields of a clinical-type fraction. Almost identical curves result if the more general equations of Montroll and Simha (9) are used. 1 hIontrol1 and Simha derived the expression tmax. = - Itmax. 01
is the length ( D P ) of the chains in which a monomeric element is most likely t o be found], where the chain length of the original material is very large and the degree of depolymerization is small. The experimentally determined number-average degrees of polymerization of the hydrolyzates corresponding t o curves B and C of Figure 5 are the same as the DP’s represented at the peak of those curves. From these considerations, and neglecting the fact that the ~ devtrans may not be linear, a dextran hydrolysis should be carried to a DPN of approximately 114, represented by the peak of curve A , Figure 5, if a maximum amount of clinical-type fraction is t o be obtained. The experimentally established fact, t h a t it is necessary t o hydrolyse to a greater extent than this, indicates that the hydrolysis of dextran cannot be depicted as being a random hydrolysis of a linear polysaccharide, all of the bonds of which are equivalent. Rather, there is an excess of reducing material compared with the statistical predictions based on assumptions of random cleavage. The deviation cannot be ascribed to the heterogeneity of the dextrans used in this work since it was previously found necessary t o hydrolyze the N R R L B-512 dextran t o approximately the same extent as indicated here for the N R R L B-1254 dextran ( 1 6 ) . This deviation is probably a measure of any one or a combination of the following:
and this occurs when the extent of cleavage, a, is denoted by the equation
Differing rates of cleavage of the several linkage types in dextran Branching in the dextran Often-noted tendency ( 1 7 ) of end bonds in a polysaccharide t o he more rapidly attacked than intermediate bonds
where n is the degree of polymerization (DP) of the degraded fragment. The yield, pn, of any fragment of a given DP at extent of hydrolysis a is given by
iontro troll and Siinha also gave a n equation (9, Equation 15) for calculating the DPw of a hydrolyzate a t known extents of depolymerization. Calculations from data obtained a t three different times during the course of the first hydrolysis listed in Table I V are as follows:
(o,L
= n a* (1
-
(3)
ay-1
Time of Hydrolysis, Hr. 1 3 4.33
0
Figure 5.
200 300 D E G R E E OF POLYMERIZATION
100
400
Theoretical Distribution of Fractions i n Dextran Hydrolyzates (7)
I n Figure 5 are shown theoretical distribution curves obtained by Equation 3. In curve A , a is the extent of hydrolysis (0.88%) which by Equation 2 would give the maximum yield of a fragment with DP of 225, the approximate number-average degree of
D P N (Found) 172 60.5 43.3
DPip (Calcd.)
DPw/DP,v
341 120
1.98 1.98 1.96
85
The values obtained for the ratio of DPw/DP*, are in accordance with the work of Sill6n ( 1 2 ) ,who pointed out that a t small values of a and with polysaccharides of initially very large D P this ratio is always approximately 2. It would be of interest a t some future time t o measure experimentally the DPw of several dextran hydrolyzate~t o check the degree of correspondence with calculated values. Finally, when the distribution of sizes in clinical-type fractions becomes better known, comparison can be made with yields theoretically t o be expected in random hydrolyses b y measurement of selected appropriate areas under the curves like those of Figure 5, either by use of the planimeter, or mathematically (IO) from the integrated form of Kuhn’s equation SUMMARY
The preparation of clinical-type fractions of dextrans synthe: sized by t h e organisms L. mesenteroides N R R L B-742 and S. deztranicum N R R L B-1254 has been accomplished in the laboratory and on a pilot plant scale of operations. The results of biological evaluation of these materials are not yet complete. I n the course of thifi work the differences among the dextrans
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
2610
studied were found to be pronounced. Conditions for the partial hydrolysis and fractionation of any particular dextran to obtain a depolymerized fraction suitable for preparing injection solutions must be established individually. Reaction rate constants were derived for hydrolysis of the dextrans. Comparison was made between the hydrolysis conditions used and those theoretically required from statistical considerations. ACKNOWLEDG-WENT
This research program on dextran has been carried out with the cooperation of a number of workers a t the Northern Utilization Research Branch. The authors wish particularly to thank V. E. Sohns and H. F. Conway for assistance in the pilot plant operations; J. C. Rankin for periodate analyses; G. E. Lauterbach for assistance in the separation of the component fractions of the K R R L B-742 dextran; C. S. \Vise for fructose analyses; Phyllis L. Patrick, C. A. Glass, and G. Gill for some of the analyses for reducing power and viscosity; C. H. VanEtten and H. F. %bel for ash, nitrogen, and heavy metals determinations; H. Davis for assistance in the hydrolysis studies; H. h l Tsuchiya, Allene Jeanes, and C. A. Wilham for supplying the high molecular weight dextrans used as raw materials for laboratory study; R. J. Dimler and F. R. Senti for helpful discussions of various phases of this work; and R. T. Milner for his keen interest which provided stimulation for these studies. LITERATURE CITED
(1) Hellman, K. N., National Research Council, Subcommittee on
Shock, and Northern Regional Research Laboratory, Peoria, Ill., “Report of Working Conference on Dextran,” p. 36, Oct. 29,1951.
Vol. 46, No. 12
Ishell, H. S.. Snyder, C. F., and associates, J . Research Natt. BUT.Standards, 50,81-6 (1953). Jeanes, A . , Haynes, W. C., and associates, J . Am. Chem. Soc., 76, in press. Jeanes, A , , Schieltz, ?;. C., and Wilham, C. A., J . B i d . Chem,., 176, 617--27 (1948). Jeanes, A , and Wilham, C. A., J. Am. Chem. SOC.,72, 2655-7 (1950). Jeanes, A., Wilham, C. A , and M i e n , J. C., J . Biol. Chem., 176, 603-15 (1948). Kuhn, W., Ber., 63, 1503-9 (1930). Lohmar, R., J . Am. Chent. SOC:,74, 4974 (1952). Montroll. E. W., and Simha, R., J . Chern. Phys., 8, 721-7 (1940). Alyrback, K., and Thorsell, W., Seensk K e m . Tidskr., 54, 50-60 (1942). Seifter, S., Dayton, S.,and associates, A r c h . Biochem., 25, 191200 (1950,.
Sillh, L. G., Siwnsk Kern. Tidskr., 55,266-79 (1943). Swanson, 11. A , , and Cori, C. F., .l.Biol. Chem., 172,797 (1948). U. S.Military Medical Purchase Description No. 4 , Sept. 19, 1952 (Stock No. 1-161-890, Dextran Injection, R I - l 6%, 500 cc., Armed Services Medical Procurement Agency, Brooklyn 1, N . Y . ) . Wise. C. S., Dimler, R. J . , and associates, presented before the Division of Carbohydrate Chemistry, 124th Meeting, B C S , Chicago, Ill., 1953. Wolff, I. A, Xehltretter, C. L., and associates, IND. Exc,. CHEM.,46,370-7 (1954). Wolfrom, AI. L., Lassettre, E. N., and O’Neill, A. N., J . Am. Chem. Soc., 73, 595-9 (1951); Jones, R. W., Dimler, R. J., and Rist, C. E., presented before the Division of Carbohydrate Chemistry, 124th Meeting, ACS, Chicago. Ill., 1953. RECEIVEDfor review April 19, 1954. ACCEPTED July 24, 1954, Presented before the Division of Carbohydrate Chemistry a t the 126th Meeting of the AMERICAN CHEMICAL SOCIETY, Kansas City, 310.
Theory of Extrusisn-Corrections I n the Symposium on Theory of Extrusion [IKD. ENG.CHEnf., 45, 970-93 (1953)j several errors occurred. The corrections printed here give the page number a t the left, followed in parentheses by the page number of the reprint booklet of the symposium. 970 (970) Equation 1 and the line preceding it, read dv/dy for
Wdv. 971 (971) second column, line 12, read read v / V for s / V .
7
for S.
I n Figure 2
972 (972) Equation 2, minus sign should be plus sign. tion 3 should read: QP
=
n h h3 cos
FP
(a
r2D2h tan ?I=-
2
(a
985 (981), bracket in numerator of Equation 7 should read:
Equa-
dz dz
980 (990), Equation 15 should read:
Equation 6 should read: QD
=
n r D N b 2 cos3 9 F D
987 (983), in Equations 2, 3, and 4 read 988 (984), Equation 17 should read:
T
for S.
973 (973), (E), date should be 1842. 977 (977) Equation 24 should read:
A h sin (a cos p 1 ~ h z s i n 2(a
-+
Q =
Equation 26 should read: sin2
(a
=
1 rDh3
m L + 2 979 (989), symbols w and 7 are not defined and definitions are not expliclt in ( 4 ) . They are defined by w =
($ +
r2D2L
In Equations 7 through 20: If the land width is not negligible-that is, if b l l is appreciably less than 1.00-dA, dZ1, and Z1 as given dl be high by the factor tlh. The diameter D in dZ1 and 2, is the mean diameter, defined in the first paper. The diameter in dZa and Z Z should be the major diameter. Major diameter equals mean diameter plus thread depth. (985)) ( 2 ) read 982 for 978. 990 (986), Equation 25 read dv/dy for ds/dy.
(988), ( 2 ) read 970 for 983.
(5),read 983 for 987.
